African Journal of Herpetology, Vol. 64, No. 2, 2015, 135–147 A comparison of DNA barcoding markers in West African frogs 1,2 3,4 HEIDI J. ROCKNEY *,CALEB OFORI-BOATENG , 1,2 1,2 NATSUKO PORCINO &ADAM D. LEACHÉ 1Burke Museum of Natural History and Culture, University of Washington, Seattle, Washington, USA; 2Department of Biology, University of Washington, Seattle, Washington, USA; 3Forestry Research Institute of Ghana, Kumasi, Ghana; 4Wildlife and Range Management Department, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana Abstract.—DNA barcoding has been proposed as a means of quick species identification using a short standardised segment of DNA. The established barcode gene for animals—the mitochondrial gene cytochrome oxidase one (CO1)—has been plagued by primer failure and low species identification success in amphibians. We investigate the accuracy of CO1 barcoding with a new dataset of West African frogs using the universal CO1 primers and new amphibian-specific CO1 primers in comparison to a proposed alternative DNA barcode for amphibians—the mitochondrial ribosomal 16s gene (16s). Research was performed using 134 specimens, comprising 21 species collected in Ghana, a global biodiversity hotspot with a deficiency of amphibian barcoding resources. These species represent 55% of amphibian species (58% of amphibian families) that are known in the area from surveys from 1988 to 2009. We found nearly a 50% increase in PCR amplification success using the amphibian-specific CO1 primers compared to the universal CO1 primers. However, the overall amplification and sequencing success of the amphibian-specific CO1 primers was low (78%) compared to the 16s gene (100%). Neither marker has a clear advantage in terms of barcoding gap; comparisons of intraspecific and interspecific variation for these markers were similar for the species we examined. Considering the qualities a barcoding gene should possess, 16s outperformed CO1 in terms of ease of obtaining sequences, and given that 16s sequences are better represented for African frogs on GenBank, this marker had higher success in BLAST searches. With amphibian species in fast decline, more consideration should be given to the appropriateness of collecting CO1 barcodes for amphibians, especially as an extensive genetic database for 16s already exists that can accurately identify amphibians. Downloaded by [98.232.95.29] at 11:29 23 December 2015 Key words.—DNA barcode; amphibians; cytochrome oxidase one (CO1); 16s rRNA; Ghana. INTRODUCTION Amphibians are an extremely endangered group of animals, with 32–41% categorised as threatened by the International Union for Conservation of Nature (IUCN 2015; Stuart et al. 2004). This is likely an underestimate as the number of described amphibian species is still increasing and there is insufficient population assessment and monitoring data to determine IUCN status on at least a third of the known amphibian species (Bickford et al. 2007; *Corresponding author. Email: [email protected] ISSN 2156-4574 print/ISSN 2153-3660 online © 2015 Herpetological Association of Africa http://dx.doi.org/10.1080/21564574.2015.1114530 http://www.tandfonline.com 136 ROCKNEY ET AL.—A comparison of DNA barcoding markers in West African frogs Fouquet et al. 2007; IUCN 2015; Köhler et al. 2005). Amphibians have morphologically different life stages, high phenotypic plasticity and high levels of cryptic diversity between species. These traits often make it difficult to correctly identify species, especially at early life-stages. DNA barcoding could assist scientists as a quick means of obtaining accurate species identifications. Collecting accurate data on a region’s biodiversity through popu- lation census and species richness is crucial to determine vulnerability status and conser- vation priority for amphibian species (Bickford et al. 2007; Fouquet et al. 2007; Hebert et al. 2004; Maya-Soriano et al. 2012; Taylor & Harris 2012; Vences et al. 2005a, b; 2012; Waugh 2007). In 2003, a barcoding system was established as a solution for rapid and accurate species identification (Hebert et al. 2003). In 2004, the Barcode of Life Database (BoLD) began compiling DNA barcodes for animal species using the only gene authorised for the animal kingdom in BoLD—a 648 bp region of the mitochondrial gene, cytochrome oxidase one (CO1). The CO1 gene has a high success rate at species identifications in numerous animal taxa including birds, fish, and many invertebrates (Hebert et al. 2003; Janzen et al. 2009; Pfenninger et al. 2007). Additionally, CO1 has shown success in identi- fying species with cryptic life stages, sexual dimorphism, and/or high phenotypic plasticity (Hebert et al. 2003; Hebert et al. 2004; Packer et al. 2009; Pfenninger et al. 2007; Alex Smith et al. 2013; Waugh 2007). However, as noted by many researchers, including DNA barcod- ing proponents, it is not realistic to expect one gene to be successful for accurate species identification across all animal taxa (DeSalle et al. 2005; Hebert et al. 2003; Janzen et al. 2009; Meier et al. 2006; Nielsen & Matz 2006; Taylor & Harris 2012; Vences et al. 2005b). Some issues that prevent the CO1 gene from being an effective barcode for all animal groups include difficulty with primer success resulting from primer site variability as well as inaccurate species identifications due to different evolutionary rates of the CO1 gene in various taxa (Davison et al. 2009; DeSalle et al. 2005; Hickerson et al. 2006; Meier et al. 2006; Meyer & Paulay 2005; Nielsen & Matz 2006; Taylor & Harris 2012; Vences et al. 2005a, b; 2012; Waugh 2007). Other problems also arise when using a single gene to accurately identify species that have high hybridisation and introgression rates, recent species divergence, or homoplasy, but this problem extends beyond just CO1 (Hebert et al. 2003; Meyer & Paulay 2005; Murphy et al. 2013; Smith et al. 2008; Taylor & Harris 2012; Vences et al. 2005 a, b; 2012; Waugh 2007). Amphibians have been one of the most problematic groups in terms of CO1 barcoding. Polymerase chain reaction (PCR) and sequencing of CO1 in amphibians has a history of Downloaded by [98.232.95.29] at 11:29 23 December 2015 low success (Davison et al. 2009; DeSalle et al. 2005; Maya-Soriano et al. 2012; Meier et al. 2006; Meyer & Paulay 2005; Smith et al. 2008; Taylor & Harris 2012; Vences et al. 2005a, b; 2012; Waugh 2007). When sequences are obtained, there is minimal success at species identification due to overlapping levels of intraspecific and interspecific variation (Fouquet et al. 2007; Maya-Soriano et al. 2012; Smith et al. 2008; Taylor & Harris 2012; Vences et al. 2005a, b). Since the barcoding movement began in 2004, CO1 barcoding of amphibians has been inundated with difficulties, and concerns have been raised whether another gene might be more suitable (Che et al. 2012; Maya- Soriano et al. 2012; Vences et al. 2005a, b). The 16s gene, which is widely used in amphi- bian systematics and taxonomy, has been proposed as an alternative DNA barcode to augment CO1 for additional confirmation of identification (Maya-Soriano et al. 2012; Vences et al. 2005a, b; 2012). However, new amphibian-specific CO1 primers, which have produced high amplification, sequencing and identification success in Malagasy Man- tellids and Asian amphibians have now raised the question of whether using 16s as a AFRICAN JOURNAL OF HERPETOLOGY 64(2) 2015 137 complementary barcoding gene is necessary for amphibian barcoding (Che et al. 2012; Jeong et al. 2013; Murphy et al. 2013; Vences et al. 2012). This research investigates the success of the new amphibian-specific primers for CO1 on species that have never been tested before. West Africa is a region with impressive amphibian biodiversity, yet minimal DNA bar- coding efforts (BoLD 2015; Ratnasingham & Hebert 2007). The Guinean rainforests in sub-Saharan Africa have been identified as a biodiversity hotspot with increasing environ- mental threats (Myers et al. 2000; Penner et al. 2011). Current rates of forest fragmentation and habitat degradation in Ghana are heavily impacting amphibian populations with one third of amphibians already considered threatened (Adum et al. 2013; Ofori-Boateng et al. 2013). Having DNA barcode profiles linked with voucher specimens will inform and assist in species monitoring and management. This area also appears to be one of the few refuges left on the planet that lacks evidence of chytrid infection and warrants close monitoring (Penner et al. 2013). In this study, we evaluate and compare the CO1 and 16s genes for use as effective bar- codes for West African amphibians. We use the universal CO1 primers, the new amphi- bian-specific CO1 primers, and the universal 16s primers on frogs collected from a fragment of the Guinean forest located in the Atewa Hills in the Eastern Region of Ghana (Fig. 1). We investigate the utility of the CO1 and 16s gene as DNA barcodes by comparing the quantity and quality of successful sequences obtained for both genes and their ability to successfully identify species. MATERIALS AND METHODS Sample and Collection We analysed 134 frog specimens from the Atewa Hills in Ghana (Table 1). The specimens were collected 26–28 May 2011. All of the specimens are archived at the Burke Museum of Natural History and Culture, University of Washington, USA. The specimens were identified in the field at the time of collection. Tissue samples were harvested and flash frozen in liquid nitrogen and subsequently stored at -80 °C. All specimens are available for loan from the Burke Museum of Natural History and Culture (accession number 2011-176). Downloaded by [98.232.95.29] at 11:29 23 December 2015 DNA Extraction, PCR Amplification and Sequencing Genomic DNA was extracted from 117 of the 134 specimens using approximately 25 mg of liver tissue with a standard salt extraction method (MacManes 2013). DNA from the other 17 specimens was extracted from skin swabs using a Qiagen kit as part of a previous study (Penner et al.